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D Nizetic

Publications and source records attributed to D Nizetic.

At least 37 records · Page 2Linked to original sources
PubMed1995

Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries.

The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and cDNA selection techniques for generating a large resource of new expressed landmarks, which contribute to the construction of a chromosome 21 transcript map.

Chromosome Mapping↗
PubMed1995

An integrated map of human chromosome 6p23.

The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich. The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23. Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region. We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA. The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region. The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs. Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets. The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side. Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.

Base Sequence↗
PubMed1994

Cloning and characterization of a 135- to 500-kb region of homology on the long arm of human chromosome 21.

The polymorphic marker D21S190 was initially isolated from a chromosome 21 phage library and mapped to two loci: one in 21q11.1 (proximal locus) and the other one in 21q22.1 (distal locus). To characterize the region of homology revealed with D21S190, we have screened two different chromosome 21 YAC libraries and one chromosome 21 cosmid library. Fluorescence in situ hybridization on normal human chromosomes of YACs, cosmids, and phages positive with D21S190 confirmed the existence of two homologous regions on the long arm of chromosome 21. Among the positive YACs, four (HY67, 2D7y21, 2D11y21, and 1B1y21) were selected and oriented relative to each other, forming a 2-Mb contig in the distal locus, including D21S54. Hybridization of YAC extremities to a panel of somatic cell hybrids containing various portions of chromosome 21 showed that the proximal locus is located between the breakpoints of 2Fu(r)1 and ACEM and the distal locus between the breakpoints of ACEM and 6918. The proximal and the distal breakpoints of JC6 are both included in the region of homology. We have constructed a restriction map of HY67, 2D7y21, and 2D11y21 spanning 1 Mb and including several markers: D21S294, D21S296, and the new STSs corresponding to YAC extremities. The region of homology encompasses 135-500 kb and has the same orientation in the distal and in the proximal locus, which are at least 12 Mb apart. It is lacking a NotI site but does contain clusters of GC-rich restriction sites, which are candidate regions for as yet unidentified genes.

Blotting, Southern↗
PubMed1994

Assignment of 55 novel cosmids to seven subregions of chromosome 13 using fluorescence in situ hybridization.

Fifty-five individual cosmids isolated from a chromosome 13-specific library have been regionally assigned using fluorescence in situ hybridization. These cosmids represent novel DNA markers that can be added to the limited number of DNA probes already available for this chromosome. Individual cosmids mapped along the length of the chromosome with almost equal distribution except that there appears to be an excess of probes from the distal 13q33-q34 region. Only two cosmids did not map back to chromosome 13, indicating that at least 96% of cosmids in this library are from this chromosome. The library is generally available as gridded colonies on nylon membranes suitable for hybridization screening.

Chromosome Walking↗
PubMed1994

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

Animals↗
PubMed1994

Efficient identification and regional positioning of YAC and cosmid clones to human chromosome 21 by radiation fusion hybrids.

The use of integrated mapping strategies involving bacterial, yeast, and rodent cells as hosts simplifies the construction of maps, which combine long-range order, high resolution, and easy access to the cloned DNA. Radiation-fusion hybrids offer a specially powerful long-range mapping system for human chromosomes. We describe here techniques for establishing a radiation-fusion hybrid map of Chromosome (Chr) 21q and its integration with local information on YAC and cosmid positions.

Animals↗
PubMed1994

Molecular cloning of a novel 11q23 breakpoint associated with non-Hodgkin's lymphoma.

Chromosomal analysis of a non-Hodgkin's lymphoma revealed a t(11;14)(q23;q32) translocation amongst other abnormalities. To investigate the molecular basis of this translocation, a cosmid library was constructed from the tumour DNA and the rearranged IGH locus was isolated in a single cosmid. Fluorescence in situ hybridization confirmed that the cloned region contained sequences from chromosome 11q23 fused to chromosome 14q32. Sequence analysis identified the breakpoint as a fusion between a region from the switch segment of the C gamma 4 gene of the IGH locus and an unknown sequence on chromosome 11. The chromosome 11 sequence maps proximal to the CD3 gene cluster and is therefore distinct from both the HTRX1 gene (rearranged in acute leukaemias) and the RCK gene (rearranged in a cell line derived from a histiocytic B-cell lymphoma). This newly identified region contains a cluster of rare cutting restriction enzyme sites located within 200 bases of the breakpoint, suggestive of a CpG island. Although this t(11;14)(q23;q32) translocation and that in the RC-K8 cell line affect different regions on chromosome 11, the breakpoints on chromosome 14 were found to have occurred at equivalent positions of S gamma 2 and S gamma 4 segments.

Adult↗
PubMed1993

High resolution cosmid and P1 maps spanning the 14 Mb genome of the fission yeast S. pombe.

Gridded on high density filters, a P1 genomic library of 17-fold coverage and a cosmid library of 8 genome equivalents, both made from S. pombe strain 972h-, were ordered by hybridizing genetic markers and individual clones from the two libraries. Yeast artificial chromosome (YAC) clones covering the entire genome were used to subdivide the libraries, and hybridization of short oligonucleotides and DNA pools made from randomly selected cosmids provided further mapping information. Restriction digests were generated as an independent confirmation of the clone order. The high resolution clone map was aligned to the genetic map and the physical Notl and YAC maps. The usefulness of the various mapping techniques and cloning procedures could be assessed upon the different data sets.

Bacteriophage P1↗
PubMed1992

Chromosomal assignment of human YAC clones by fluorescence in situ hybridization: use of single-yeast-colony PCR and multiple labeling.

Alu-PCR provides a convenient tool for amplification of human-specific sequences from yeast DNA containing yeast artificial chromosomes (YAC) clones. PCR products can be labeled nonisotopically and hybridized in situ, and the chromosomal origin of the clones can be determined. This avoids time-consuming gel purification of the yeast artificial chromosome and the low-efficiency procedure of labeling whole yeast DNA containing the YAC. The application of Alu-PCR to single-yeast colonies permits the mapping of YACs at a very early stage of their characterization. In situ hybridization can detect clones with noncontiguous fragments of DNA, and these can be discarded without further time-consuming characterization. To increase further the potential of the method, we show the application of multicolor hybridization techniques.

Base Sequence↗
PubMed1991

W (A or T) sequences as probes and primers suitable for genomic mapping and fingerprinting.

A limitation to the use of oligonucleotide probes as tools for genetic and physical mapping has been the low hybridization positive frequency obtained by oligonucleotides of sufficient length to hybridize preferentially to cloned insert DNA (and not host E. coli genomic DNA). Both computer and experimental results now indicate that oligonucleotide probes composed of W (A or T) sequence are preferentially found in eukaryotic DNA, and can be used to provide high frequency, discriminative hybridization. Such W sequences may be useful as either probes or PCR primers in molecular diagnostic applications as well as in genetic and physical mapping.

Adenine↗
PubMed1991

Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products.

A direct hybridization protocol is described for screening cosmid and yeast artificial chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual Alu-PCR product before library screening. A series of human X chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis.

Base Sequence↗
PubMed1990

Ordering of cosmid clones covering the herpes simplex virus type I (HSV-I) genome: a test case for fingerprinting by hybridisation.

To allow the efficient construction of ordered clone libraries, we have been investigating the use of 'oligonucleotide fingerprinting' as an approach to identify overlapping clones, and ultimately restore the linear order of the clone set. To test the effectiveness of the procedure, we have constructed a cosmid library from the genome of the human DNA virus HSV-I and used hybridisation to multiple oligonucleotides selected from the nucleotide sequence to reconstruct the order of clones and oligonucleotides on the genome.

Base Sequence↗